Isolation and Confirmation of Yersinia pestis Mutants Exempt from Select Agent Regulations

Robert D. Perry1, Scott W. Bearden2

1 Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky, 2 Centers for Disease Control and Prevention, Division of Vector‐Borne Infectious Diseases, Fort Collins, Colorado
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 5B.2
DOI:  10.1002/9780471729259.mc05b02s11
Online Posting Date:  November, 2008
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This unit describes protocols for Yersinia pestis to confirm plasmid profiles, construct and confirm a Δpgm mutation, and cure the low‐calcium response (Lcr) plasmid encoding a type III secretion system (TTSS). Strains lacking either the chromosomal pgm locus or the Lcr plasmid can be safely studied under BSL‐2 conditions and are exempt from Select Agent regulations in the U.S. Curr. Protoc. Microbiol. 11:5B.2.1‐5B.2.12. © 2008 by John Wiley & Sons, Inc.

Keywords: plague; plasmid isolation; low‐calcium response; type III secretion; pigmentation; biofilm

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolation of Plasmid DNA
  • Alternate Protocol 1: Rapid Lysis Plasmid Isolation
  • Basic Protocol 2: Isolation and Demonstration of a pCD‐Negative Strain
  • Basic Protocol 3: Isolation and Demonstration of a Δpgm Mutation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Isolation of Plasmid DNA

  • Y. pestis liquid culture
  • B & D solution 1 (see recipe), ice cold
  • B & D solution 2 (see recipe)
  • B & D solution 3 (see recipe), ice cold
  • Phenol/CHCl 3 solution (see recipe)
  • 95% ethanol
  • 1× TE‐RNase (see recipe)
  • 10× DNA loading buffer (see recipe)
  • DNA size standard
  • 0.7% (w/v) agarose gel
  • 1× TBE running buffer ( appendix 2A)
  • Gel‐Star stain (use according to manufacturer's instructions) or 1% ethidium bromide solution
  • 1 M NaCl
  • 1.5‐ to 2.0‐ml microcentrifuge tubes
  • Electrophoresis gel box
  • Electrophoresis power supply
  • UV light box
  • Gel photographic equipment

Alternate Protocol 1: Rapid Lysis Plasmid Isolation

  • 18‐ to 24‐hr Y. pestis culture on SBA or TBA plate
  • TE buffer ( appendix 2A)
  • Kado lysis buffer (see recipe)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 2A)
  • 0.6% SeaKem Gold agarose gel in 1× TBE (or other high‐quality agarose)
  • Inoculating loop, sterile
  • 60°C heating block or water bath
  • Refrigerated microcentrifuge

Basic Protocol 2: Isolation and Demonstration of a pCD‐Negative Strain

  • Y. pestis strain
  • Heart infusion broth (HIB, Difco)
  • TBA‐MgOX plates (see recipe)
  • CR plates (see recipe in unit 5.1)
  • 37°C incubator
  • Inoculating loop, sterile

Basic Protocol 3: Isolation and Demonstration of a Δpgm Mutation

  • Y. pestis strain
  • Heart infusion broth (HIB, Difco)
  • 33 mM K‐PO 4 buffer
  • CR plates (see recipe in unit 5.1)
  • 25 mM MgCl 2
  • 10× dNTP stocks (2 mM each of dATP, dCTP, dGTP, and dTTP)
  • Taq polymerase and 10× buffer
  • 10× DNA loading dye
  • DNA standards
  • 1% (w/v) agarose gel
  • Ethidium bromide
  • 26° to 30°C incubator
  • Spectrophotometer
  • Dilution tubes
  • DNA thermal cycler
  • Electrophoresis power supply
  • Electrophoresis gel box
  • UV light box
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Literature Cited

Literature Cited
   Anisimov, A.P., Dentovskaya, S.V., Titareva, G.M., Bakhteeva, I.V., Shaikhutdinova, R.Z., Balakhonov, S.V., Lindner, B., Kocharova, N.A., Senchenkova, S.N., Holst, O., Pier, G.B., and Knirel, Y.A. 2005. Intraspecies and temperature‐dependent variations in susceptibility of Yersinia pestis to the bactericidal action of serum and to polymyxin B. Infect. Immun. 73:7324‐7331.
   Bearden, S.W., Fetherston, J.D., and Perry, R.D. 1997. Genetic organization of the Yersiniabactin biosynthetic region and construction of avirulent mutants in Yersinia pestis. Infect. Immun. 65:1659‐1668.
   Birnboim, H.C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513‐1523.
   Brubaker, R.R. 1969. Mutation rate to nonpigmentation in Pasteurella pestis. J. Bacteriol. 98:1404‐1406.
   Brubaker, R.R. 2005. Influence of Na+, dicarboxylic amino acids, and pH in modulating the low‐calcium response of Yersinia pestis. Infect. Immun. 73:4743‐4752.
   Fetherston, J.D., Schuetze, P., and Perry, R.D. 1992. Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element. Mol. Microbiol. 6:2693‐2704.
   Fetherston, J.D., Bertolino, V.J., and Perry, R.D. 1999. YbtP and YbtQ: Two ABC transporters required for iron uptake in Yersinia pestis. Mol. Microbiol. 32:289‐299.
   Geoffroy, V.A., Fetherston, J.D., and Perry, R.D. 2000. Yersinia pestis YbtU and YbtT are involved in synthesis of the siderophore Yersiniabactin but have different effects on regulation. Infect. Immun. 68:4452‐4461.
   Inglesby, T.V., Dennis, D.T., Henderson, D.A., Bartlett, J.G., Ascher, M.S., Eitzen, E., Fine, A.D., Friedlander, A.M., Hauer, J., Koerner, J.F., Layton, M., McDade, J., Osterholm, M.T., O'Toole, T., Parker, G., Perl, T.M., Russell, P.K., Schoch‐Spana, M., and Tonat, K. 2000. Plague as a biological weapon: Medical and public health management. J. Amer. Med. Assoc. 283:2281‐2290.
   Jones, H.A., Lillard, J.W. Jr., and Perry, R.D. 1999. HmsT, a protein essential for expression of the haemin storage (Hms+) phenotype of Yersinia pestis. Microbiology 145:2117‐2128.
   Kado, C.I. and Liu, S.‐T. 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 45:1365‐1373.
   Kirillina, O., Fetherston, J.D., Bobrov, A.G., Abney, J., and Perry, R.D. 2004. HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms‐dependent biofilm formation in Yersinia pestis. Mol. Microbiol. 54:75‐88.
   Kutyrev, V.V., Protsenko, O.A., Smirnov, G.B., Boolgakova, E., Kukleva, L.M., Zudina, I.V., Vidyaeva, N.A., and Koozmichenko, I. 2003. Yersinia pestis from natural foci. Adv. Exp. Med. Biol. 529:313‐316.
   Lathem, W.W., Crosby, S.D., Miller, V.L., and Goldman, W.E. 2005. Progression of primary pneumonic plague: A mouse model of infection, pathology, and bacterial transcriptional activity. Proc. Natl. Acad. Sci. U.S.A. 102:17786‐17791.
   Perry, R.D., Pendrak, M.L., and Schuetze, P. 1990. Identification and cloning of a hemin storage locus involved in the pigmentation phenotype of Yersinia pestis. J. Bacteriol. 172:5929‐5937.
   Perry, R.D. and Fetherston, J.D. 1997. Yersinia pestis—Etiologic agent of plague. Clin. Microbiol. Rev. 10:35‐66.
   Straley, S.C. and Starnbach, M.N. 2000. Yersinia: Strategies that thwart immune defenses. In Effects of Microbes on the Immune System (M.W. Cunningham and R.S. Fujinami, eds.) pp. 71‐92. Lippincott Williams & Wilkins, Philadelphia.
   Surgalla, M.J. and Beesley, E.D. 1969. Congo red‐agar plating medium for detecting pigmentation in Pasteurella pestis. Appl. Microbiol. 18:834‐837.
   Une, T. and Brubaker, R.R. 1984. In vivo comparison of avirulent Vwa– and Pgm− or Pstr phenotypes of Yersiniae. Infect. Immun. 43:895‐900.
   Welkos, S., Pitt, M.L.M., Martinez, M., Friedlander, A., Vogel, P., and Tammariello, R. 2002. Determination of the virulence of the pigmentation‐deficient and pigmentation‐/plasminogen activator‐deficient strains of Yersinia pestis in non‐human primate and mouse models of pneumonic plague. Vaccine 20:2206‐2214.
   Zahorchak, R.J. and Brubaker, R.R. 1982. Effect of exogenous nucleotides on Ca2+ dependence and V antigen synthesis in Yersinia pestis. Infect. Immun. 38:953‐959.
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